Biologically active neopeltolide compounds

ABSTRACT

The subject invention provides novel compositions of biologically active macrolide compounds which can advantageously be used in blocking cellular proliferation, treatment of cancer, treatment of fungal infections and control of spoilage of food, cosmetic and other consumer items.

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisionalapplication Ser. No. 60/482,216, filed Jun. 23, 2003.

FIELD OF THE INVENTION

[0002] This invention relates to organic compounds and compositionswhich have useful therapeutic properties. More particularly, theinvention concerns novel macrolide compounds having anti-proliferative,antitumor and antifungal activities; Pharmaceutical compositionscomprising such compounds; and methods of their use for therapeuticpurposes and in the control of spoilage of food, cosmetic and otherconsumer items.

BACKGROUND OF THE INVENTION

[0003] Of great importance to man is the control of pathologicalcellular proliferation such as that which occurs in the case of cancer.Considerable research and resources have been devoted to oncology andantitumor measures including chemotherapy. While certain methods andchemical compositions have been developed which aid in inhibiting,remitting, or controlling the growth of, for example, tumors, newmethods and antitumor chemical compositions are needed.Anti-proliferative agents can also be useful in treating autoimmunediseases and inflammatory disease.

[0004] In searching for new biologically active compounds, it has beenfound that some natural products and organisms are potential sources forchemical molecules having useful biological activity of great diversity.For example, the diterpene commonly known as paclitaxel, isolated fromseveral species of yew trees, is a mitotic spindle poison thatstabilizes microtubules and inhibits their depolymerization to freetubulin (Fuchs, D. A., R. K. Johnson [1978] Cancer Treat. Rep.62:1219-1222; Schiff, P. B., J. Fant, S. B. Horwitz [1979] Nature(London) 22:665-667). Paclitaxel is also known to have antitumoractivity and has undergone a number of clinical trials which have shownit to be effective in the treatment of a wide range of cancers(Rowinski, E. K. R. C. Donehower [1995] N. Engl. J. Med. 332:1004-1014).See also, e.g., U.S. Pat. Nos. 5,157,049; 4,960,790; and 4,206,221.

[0005] Marine sponges have also proven to be a source of biologicallyactive chemical molecules. A number of publications disclose organiccompounds derived from marine sponges including Scheuer, P. J. (ed.)Marine Natural Products, Chemical and Biological Perspectives, AcademicPress, New York, 1978-1983, Vol. I-V; Uemura, D., K. Takahashi, T.Yamamoto, C. Katayama, J. Tanaka, Y. Okumura, Y. Hirata (1985) J. Am.Chem. Soc. 107:4796-4798; Minale, L. et al. (1976) Fortschr. Chem. org.Naturst. 33:1-72 Faulkner, D. J., Nat. Prod. Reports 1984, 1, 251-551;ibid. 1987, 4, 539; ibid 1990, 7, 269; ibid 1993, 10, 497; ibid 1994,11, 355; ibid 1995, 12, 22; ibid 1998, 15:113-58; ibid 2000 17:1-6; ibid2000 17: 7-55; ibid 2001, 18: 1-49; 2002, 19: 1-48.; Gunasekera, S. P.,M. Gunasekera, R. E. Longley and G. K. Schulte (1990) J. Org. Chem.,55:4912-4915.; Horton, P. A., F. E. Koehn, R. E. Longley, and O. J.McConnell, (1994) J. Am. Chem. Soc. 116: 6015-6016.

[0006] The success of chemotherapy for the treatment of various cancerscan be substantially negated though cellular mechanisms which haveevolved to enable neoplastic cells to subvert the cytotoxic effects ofthe drug. Some cells have developed mechanisms, which confer resistanceto a number of structurally unrelated drugs. This multi-drug resistance(or MDR) phenomenon may arise through a number of different mechanisms.One of these involves the ability of a cell to reduce intracellularconcentrations of a given drug through efflux from cytoplasm through andout the cell membrane by a series of unique ATP-dependent transporterproteins called-P-glycoproteins (Pgp) (Casazza, A. M. and C. R.Fairchild [1996] “Paclitaxel (Taxol®): mechanisms of resistance” CancerTreat Res. 87:149-171). The surface membrane, 170 kDa Pgp, is encoded bythe mdr-1 gene and appears to require substrate binding before transportbegins. A wide range of compounds, including a number of structurallyunrelated chemotherapeutic agents (adriamycin, vinblastine, colchicine,etoposide and Taxol), are capable of being transported by Pgp and renderthe cell resistant to the cytotoxic effects of these compounds. Whilemany normal cell types possess Pgp, in general, tumor cell lines, whichpossess high levels of mRNA specific for Pgp, also exhibitoverexpression of membrane Pgp and demonstrate resistance to variousdrugs. This intrinsic resistance can be increased multifold byincubation of cells with stepwise increasing doses of a particular drugover a period of several months. This can be further facilitated by theaddition of the MDR reversal agent, verapamil (Casazza, A. M. and C. R.Fairchild [1996] supra) in combination with the particular drug. Drugresistant cell lines produced in this fashion exhibit resistance to drugcytotoxicity from 20 to 500 fold, compared to parental cell lines.

[0007] An additional target for cancer drug discovery is a highmolecular weight membrane protein associated with multi-drug resistanceproperties of certain tumor cells known as the multidrugresistance-associated protein (MRP). MRP is a 190 kD membrane-boundglycoprotein (Bellamy, W. T. [1996], Annu. Rev. Pharmacol. Toxicol., 36:161-183.) which belongs to the same family of proteins as thep-glycoprotein pump P-gp (Broxterman, H. J., Giaccone, G., and Lankelma,J. [1995], Current Opinion in Oncology, 7:532-540.) but shares less than15% homology of amino acids with P-gp (Komorov, P. G., Shtil, A. A.,Holian, O., Tee, L., Buckingham, L., Mechetner, E. B., Roninson, I. B.,and Coon, J. S. [1998], Oncology Research, 10: 185-192.). MRP has beenfound to occur naturally in a number of normal tissues, including liver,adrenal, testis, and peripheral blood mononuclear cells (Krishan, A.,Fitz, C. M., and Andritsch, I. [1997], Cytometry, 29: 279-285). MRP hasalso been identified in tissues of the lung, kidney, colon, thyroid,urinary bladder, stomach, spleen (Sugawara, I. [1998] The CancerJournal, 8(2) and skeletal muscle (Kruh, G. D., Gaughan, K. T., Godwin,A., and Chan, A. [1995], Journal of the National Cancer Institute,87(16): 1256-1258.). High levels of MRP have been implicated inmultidrug resistance (MDR) in cancers of the lung and pancreas (Miller,D. W., Fontain, M., Kolar, C., and Lawson, T. [1996]. Cancer Letters,107: 301-306.), and in neuroblastomas, leukemias and cancer of thethyroid (Kruh, G. D., Gaughan, K. T., Godwin, A., and Chan, A. [1995],Journal of the National Cancer Institute, 87(16): 1256-1258.), as wellas bladder, ovarian and breast cancers (Barrand, M., Bagrij, T., andNeo, S. [1997]., General Pharmacology, 28(5): 639-645.). MRP-mediatedMDR involves some of the same classes of compounds as those which aremediated by P-gp, including vinca alkaloids, epipodophyllotoxins,anthracyclins and actinomycin D (Barrand, M., Bagrij, T., and Neo, S.[1997]., General Pharmacology, 28(5): 639-645). However, the substratespecificity has been demonstrated to differ from that of P-gp (Komorov,P. G., Shtil, A. A., Holian, O., Tee, L., Buckingham, L., Mechetner, E.B., Roninson, I. B., and Coon, J. S. [1998], Oncology Research, 10:185-192.). Drugs which would inhibit or which are not substrates for theMDR pump would, therefore, be useful as chemotherapeutic agents.

[0008] Of further significant importance to man is the control of fungiwhich can cause human, animal and plant diseases as well as food andproduct spoilage. Considerable research and resources have been devotedto identifying antifungal agents. While certain methods and chemicalcompositions have been developed that aid in inhibiting or controllingthe growth of fungi, new methods and antifungal compositions are needed.

[0009] Human mycotic infections may be grouped into superficial,subcutaneous, and deep (or systemic) mycoses. Superficial fungalinfections of skin, hair and nails may be chronic and resistant totreatment but rarely affect the general health of the patient. Deepmycoses, on the other hand, may produce systemic involvement and aresometimes fatal.

[0010] The deep mycoses are caused by organisms that live free in naturein soil or on decaying organic material and are frequently limited tocertain geographic areas. In such areas, many people acquire the fungalinfection. A majority develop only minor symptoms or none at all, andonly a small minority of infections progress to full-blown serious orfatal disease. The host's cell-mediated immune reactions are ofparamount importance in determining the outcome of such infections.

[0011] Post-harvest losses during storage of plant produce are caused,interalia, by fungal and bacterial pathogens. Fungicidal compounds havelong been used to increase yields and extend agricultural productioncapabilities into new areas. They have also been extremely importanttools for ameliorating season-to-season differences in yield and qualitycaused by weather-driven variations in disease pressure.

[0012] Chemical fungicides have provided an effective method of control;however, the public has become concerned about the amount of residualchemicals which might be found in food, ground water and theenvironment. Stringent new restrictions on the use of chemicals and theelimination of some effective pesticides from the market place couldlimit economical and effective options for controlling fingi.

[0013] One example of the need to control post-harvest spoilage ofagriculture products pertains to green and blue molds of citrus fruitscaused by Penicillium digitatum and P. italicum. These molds causesevere damage during storage and shipping. The existing fresh-marketindustry relies completely on a combination of several chemicaltreatments to deliver sound fruit to distant markets over substantialperiods of time without excessive damage caused by these molds.Unfortunately, there are increasing concerns about the safety of thechemicals currently used to control these fungal pathogens. Also, thereare increasing problems with fungal strains with resistance to the mosteffective compounds.

[0014] In another example, powdery mildew of grapes caused by Uncinulanecator can cause severe damage even in dry areas such as California.Traditionally this disease was controlled with applications of elementalsulfur, but this necessitates frequent, high volume applications of anirritating material. The introduction of ergosterol biosynthesisinhibiting fungicides (primarily triazoles) greatly simplifies control,but also selects for tolerant strains. Some of these compounds are alsoknown to have potential teratogenic effects and very long soilresiduals. In these and other examples, alternative control methods arein great demand—particularly methods which are safer or moreenvironmentally benign.

[0015] To prevent fungal spoilage it is common practice in manycountries to spray produce with systemic fungicides in the field and todip harvested produce in fungicide solutions prior to storage. Since theoncogenic nature of many of the most commonly used fungicides isincreasingly recognized and because the persistence of most fungicidesis increased by the low storage temperatures the postharvest use offungicides is of growing concern.

[0016] Additionally, resistance to the fungicides, used has beenreported and suppression of the main spoilage organism B. cinera byfungicides such as BENOMYL fungicide has been shown to result inincreased population of A. brassicicola which causes a more penetratingrot of produce than B. cinera. See, for example, U.S. Pat. No.5,869,038.

[0017] The future role of fungicides in agriculture is increasinglythreatened by several factors including; the development of pestresistance, increasing concerns about food safety and environmentalaccumulation of toxic compounds. As older fungicides are removed fromthe market due to regulatory changes there is an increasing need to findnew effective fungicidal compounds.

BRIEF SUMMARY OF THE INVENTION

[0018] The subject invention provides novel compositions of biologicallyactive compounds that have utility for use in inhibiting cellularproliferation. In a specific embodiment, the compounds and compositionsof the subject invention can be used in the treatment of cancer. In asecond specific embodiment, the compounds and compositions of thesubject invention can be used in the control of fungal growth.

[0019] In one embodiment, the novel compositions and methods of thesubject invention can be used in the treatment of an animal hostingcancer cells including, for example, inhibiting the growth of tumorcells in a mammalian host. More particularly, the subject compounds canbe used for inhibiting in a human the growth of tumor cells, includingcells of breast, colon, CNS, ovarian, renal, prostate, liver,pancreatic, uterine, or lung tumors, as well as human leukemia ormelanoma cells. The mechanisms for achieving anticancer activityexhibited by the subject compounds would lead a person of ordinary skillin the art to recognize the applicability of the subject compounds,compositions, and methods to additional types of cancer as describedherein.

[0020] In accordance with the subject invention, methods for inhibitingcancer cells in a host include contacting tumor cells with an effectiveamount of the new pharmaceutical compositions of the invention. Thecancer cells inhibited by the invention are those which are susceptibleto the subject compounds described herein or compositions comprisingthose compounds.

[0021] In a further specific embodiment, the novel compositions andmethods of the subject invention subject can be used in the control offungal growth. Because of the biological activity of these compounds,they can be used for treatment of plant and animal fungal infections, toprevent spoilage of organic compositions such as food and cosmetics, andas disinfectants. In a preferred embodiment, the novel compounds,compositions and methods of use of the subject invention canadvantageously be used to inhibit the growth of fungi in a mammalianhost. As used herein, reference to “antifungal activity” includesfungicidal and fungistatic activity as well as the inhibition of fungalgermination or growth.

[0022] In specific embodiments, the subject invention provides newmacrolides, as exemplified by Neopeltolide (I).

[0023] Neopeltolide has not been isolated previously from a naturalsource nor has it been previously synthesized.

[0024] Additional aspects of the invention include the provision ofmethods for producing the new compounds and compositions.

[0025] Other objects and further scope of applicability of the presentinvention will become apparent from the detailed descriptions givenherein; it should be understood, however, that the detaileddescriptions, while indicating preferred embodiments of the invention,are given by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent from such descriptions.

DETAILED DISCLOSURE OF THE INVENTION

[0026] The subject invention provides novel compositions of biologicallyactive macrolide compounds which are useful for inhibiting pathologicalcellular proliferation.

[0027] Advantageously, the macrolide compounds of the subject inventioncan be used to inhibit unwanted cellular proliferation, including thepathogenic proliferation of tumor and fungal cells.

[0028] In a preferred embodiment, these compounds can be used fortreating cancer. More specifically, the novel compounds, compositionsand methods of use can advantageously be used to inhibit the growth oftumor and other cancer cells in a mammalian host. As described herein,the compounds of the subject invention have utility for use in thetreatment of cancer. More particularly, the subject compounds can beused for inhibiting in a human the growth of tumor cells, includingcells of breast, prostate, colon, CNS, ovarian, renal, liver,pancreatic, uterine, or lung tumors, as well as human leukemia ormelanoma cells. The compounds also have utility in the treatment ofmulti-drug resistant cancer cells.

[0029] The subject invention further provides novel compositions ofbiologically active macrolide compounds which are useful in the controlof fungal growth. Because of the biological activity of these compounds,they can be used for treatment of plant and animal fungal infections, toprevent spoilage of organic compositions such as food and cosmetics, andas disinfectants. In a preferred embodiment, the novel compounds,compositions and methods of use of the subject invention canadvantageously be used to inhibit the growth of fungi in a mammalianhost. As used herein, reference to “antifungal activity” includesfungicidal and fungistatic activity as well as the inhibition of fungalgermination or growth.

[0030] In a preferred embodiment, the subject invention providescompounds having the following formula:

[0031] In a further specific embodiment, the subject invention providescompounds having the following formula

[0032] The subject invention further pertains to isolated enantiomericcompounds. The isolated enantiomeric forms of the compounds of theinvention are substantially free from one another (i.e., in enantiomericexcess). In other words, the “R” forms of the compounds aresubstantially free from the “S” forms of the compounds and are, thus, inenantiomeric excess of the “S” forms. Conversely, “S” forms of thecompounds are substantially free of “R” forms of the compounds and are,thus, in enantiomeric excess of the “R” forms. In one embodiment of theinvention, the isolated enantiomeric compounds are at least about in 80%enantiomeric excess. In a preferred embodiment, the compounds are in atleast about 90% enantiomeric excess. In a more preferred embodiment, thecompounds are in at least about 95% enantiomeric excess. In an even morepreferred embodiment, the compounds are in at least about 97.5%enantiomeric excess. In a most preferred embodiment, the compounds arein at least about 99% enantiomeric excess.

[0033] In accordance with the subject invention, methods for inhibitingcancer in a host include contacting cancer cells with an effectiveamount of the new pharmaceutical compositions of the invention. Thetumor cells inhibited by the invention are those which are susceptibleto the subject compounds described herein or compositions comprisingthose compounds.

[0034] The subject invention further provides methods of use of the newcompounds and compositions of the invention, e.g., methods of inhibitingtumors and other cancer cells in an animal, preferably a mammal. Mostpreferably, the invention comprises a method for the antitumor treatmentof a human in need of such treatment, i.e., a human hosting cancercells, including breast, colon, liver, pancreatic, uterine, or lungtumor cells, or leukemia cells including multi-drug resistant cancercells.

[0035] In preferred embodiments of the invention, the compounds aresubstantially pure, i.e., contain at least 95% of the compound asdetermined by established analytical methods.

[0036] In further preferred methods of the invention, salts and analogswithin the scope of the invention are made by adding mineral acids,e.g., HCl, H₂SO₄, or strong organic acids, e.g., formic, oxalic, inappropriate amounts to form the acid addition salt of the parentcompound or its derivative. Also, synthesis type reactions may be usedpursuant to known procedures to add or modify various groups in thepreferred compounds to produce other compounds within the scope of theinvention.

[0037] As used in this application, the terms “analogs,” refers tocompounds which are substantially the same as another compound but whichmay have been modified by, for example, adding or removing side groups.

[0038] The scope of the invention is not limited by the specificexamples and suggested procedures and uses related herein sincemodifications can be made within such scope from the informationprovided by this specification to those skilled in the art.

[0039] A more complete understanding of the invention can be obtained byreference to the following specific examples of compounds, compositions,and methods of the invention. The following examples illustrateprocedures for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted. It will beapparent to those skilled in the art that the examples involve use ofmaterials and reagents that are commercially available from knownsources, e.g., chemical supply houses, so no details are givenrespecting them.

EXAMPLE 1 Isolation and Structure Elucidation of Neopeltolide (I)

[0040] A. Collection and Taxonomy of the Source Organism

[0041] A sample of a sponge identified as belonging to the FamilyNeopeltidae (Phylum: Porifera, Class Demospongiae, Order Lithistida,Family Neopeltidae) was collected using the Johnson Sea Link mannedsubmersible at a depth of 433 m off the North Jamaican Coast (latitude18 28.638′N, longitude 78 10.996′W). The sponge morphology is that of aplate with visible pores on the upper surface, stony in consistency, andwhite in color. A reference sample preserved in ethanol has beendeposited in the Harbor Branch Oceanographic Museum (catalog number003:01004, DBMR number 23-VIII-93-5-010) and is available for taxonomicevaluation by those skilled in the art.

[0042] B. Isolation and Structure Elucidation of Neopeltolide (I).

[0043] One hundred and five (105) grams of the frozen Neopeltidaesponge, 23-VIII-93-5-010 was extracted exhaustively by macerating withethanol using a Waring Blender (5×250 mL). The combined filteredextracts were concentrated by distillation under reduced pressure toyield 3.2 g of crude residue. The residue was partitioned betweenn-butanol and water (3×50 ml portions). After concentration, then-butanol phase (0.7 g) was chromatographed under vacuum columnchromatographic conditions on an Kieselgel 60H (EM SCIENCE) stationaryphase. A 150 mL Buchner funnel fitted with a medium porosity frittedglass disc was used as the column. The stationary phase was packed to atotal height of 4 cm. The butanol partition was applied as a slurry tothe column in a mixture of heptane-ethyl acetate (8:2 v/v).

[0044] Fractions were eluted using a 20% step gradient of ethyl acetatein heptane followed by a series of fractions containing increasingamounts of methanol in ethyl acetate [Fraction 1: heptane-ethyl acetate80:20 v/v (150 ml); Fraction 2: heptane-ethyl acetate 60:40 v/v (150ml); fraction 3: heptane-ethyl acetate 40:60 v/v (1150 ml); Fraction 4:heptane-ethyl acetate 25:75 v/v (150 ml); Fraction 5: heptane-ethylacetate 20:80 v/v (150 ml); Fraction 6: ethyl acetate (150 ml); Fraction7: ethyl acetate-methanol 75:25 v/v (150 ml); Fraction 8: ethylacetate-methanol 50:50 v/v (150 ml); Fraction 9 methanol (150 ml).

[0045] Neopeltolide eluted cleanly into Fraction 3. Fraction 3 wasfurther separated by HPLC using a Vydac C-18 Protein and Peptide Column(10 mm×250 mm, 10μ particle size), flow rate=3 ml/min; eluent:water:acetonitrile 40:60 v/v; monitored by UV at 230 nm. Neopeltolideelutes after approximately 6.2 column volumes under these conditions.

[0046] Neopeltolide (I): colorless oil; MS: m/z observed 591.32692,calculated 591.32816 Δ=−1.2 mmu for formula C₃₁H₄₇O₉N₂ (M+H⁺); [α]²⁴_(D)=+23.0 (c 0.0024 g/ml in methanol). See Table 1 for ¹H and ¹³C NMRdata. TABLE 1 NMR Data for Neopeltolide (I) Atom # ¹H (mult. J in Hz)¹³C (mult)  1 — 172.5 (s)  2 A 2.66 (dd J = 14.8, 4.3)  43.0 (t) B 2.33(dd J = 14.5, 10.8)  3 4.09 (m)  70.6 (d)  4 A 1.84 (m)  35.9 (t) B 1.53(m)  5 5.21 (m)  68.5 (d)  6 A 1.68 (m)  37.0 (t) B 1.54 (m)  7 3.58 (ddJ = 10.4, 9.8)  76.6 (d)  8 A 1.40 (m)  44.8 (t) B 1.29 (m)  9 1.45 (m) 32.0 (d) 10 A 1.58 (m)  42.9 (t) B 1.14 (m) 11 3.63 (m)  76.5 (d) 12 A1.90 (m)  40.5 (t) B 1.39 (m) 13 5.18 (m)  73.8 (d) 14 A 1.75 (m) B 1.53(m)  37.5 (t) 15 AB 1.39 (2H m)  19.6 (t) 16 0.93 (3H t J = 7.3)  14.1(q) 17 0.99 (3H d J = 8.8)  25.9 (q) 11-OCH3 3.33 (3H s)  56.4 (q)  1′ —166.4 (s)  2′ 5.91 (bd J = 11.3) 121.3 (d)  3′ 6.35 (dt J = 11.3, 7.45)149.8 (d)  4′ AB 3.02 (2H q J = 7.45)  28.4 (t)  5′ AB 2.73 (2H t J =7.45)  26.0 (t)  6′ — 141.5 (s)  7′ 7.55 (bs) 135.3 (d)  8′ — 161.3 (s) 9′ 6.26 (bd J = 12.0) 115.8 (d) 10′ 6.05 (ddd J = 12.0, 6.0, 6.0) 138.5(d) 11′ AB 4.31 (2H bd J = 4.9)  40.6 (t) 12′ 158.9 (s) 12′-OCH3 3.67(3H s)  52.4 (q)

EXAMPLE 2 Antitumor Effects of Neopeltolide (I)

[0047] A. Effects of Neopeltolide on In Vitro Proliferation of TumorCell Lines

[0048] Neopeltolide was analyzed as to its effect on the proliferationof A549 human lung adenocarcinoma, NCI-ADR-RES (Formerly MCF-7/ADR)human breast cancer and P388 murine leukemia cell lines. P388 cells wereobtained from Dr. R. Camalier, National Cancer Institute, Bethesda, Md.,and A549 and NCI-ADR-RES cells were obtained from American Type CultureCollection, Rockville, Md.

[0049] All cell lines are maintained in Roswell Park Memorial Institute(RPMI) medium 1640 supplemented with 100 U/mL penicillin 100 μg/mlstreptomycin, 60 μg/ml L-glutamine, 18 mM HEPES, 0.05 mg/mL gentamycinand 10% fetal bovine serum. Cell lines are cultured in plastic tissueculture flasks and kept in an incubator at 37° C. in humidified aircontaining 5% CO₂.

[0050] To assess the antiproliferative effects of agents against thevarious cell lines, 200 μl cultures (96-well tissue culture plates,Nunc, Denmark) are first established at 3×10⁴ cells/ml for adherentlines (A549, NCI ADR-RES) and 1×10⁵ for non-adherent lines (P388) intissue culture medium and incubated for 24 hr at 37° C. in 10% CO₂ inair in order to allow cells to attach. A volume of 100 μl of medium isremoved from each test well and 100 μl of medium containing serial,two-fold dilutions of the test agent is added to each well containingtumor cells. Medium without drug is also added to wells containing tumorcells which serve as no drug controls. Positive drug controls areincluded to monitor drug sensitivity of each of the cell lines. Theseinclude varying dilutions of 5-fluorouracil, doxorubicin.

[0051] After 72-h exposures (Adherent cell lines) or 48-hr exposure(Non-adherent cell lines), tumor cells are enumerated using3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (M.C. Alley, et al., Cancer Res. 48:589, 1988) as follows:

[0052] A volume of 75 μl of warm growth media containing 5 mg/ml MTT isadded to each well, cultures returned to the incubator, and leftundisturbed for 3 hours. To spectrophotometrically quantitate formationof reduced formazan, plates are centrifuged (900×g, 5 minutes), culturefluids removed by aspiration, and 200 μl of acidified isopropanol (2 mlconcentrated HC/liter isopropanol) added per well. The absorbance of theresulting solutions is measured at 570 nm with a plate reader (SpectraII Tecan Laboratories).

[0053] The absorbance of tests wells is divided by the absorbance ofdrug-free wells, and the concentration of agent that results in 50% ofthe absorbance of untreated cultures (IC₅₀) is determined by linearregression of logit-transformed data (D. J. Finney, Statistical Methodin Biological Assay, third ed., pp.316-348, Charles Griffin Co., London,1978). A linear relationship between tumor cell number and formazanproduction has been routinely observed over the range of cell densitiesobserved in these experiments.

[0054] The two standard drug controls (indicated above) are included ineach assay to monitor the drug sensitivity of each of the cell lines andIC₅₀ values are determined for each drug-cell combination.

[0055] A summary of results in these assays for Neopelotide I can befound in Table 2. TABLE 2 Cytotoxicity Results for Neopeltolide A549NCIADR-RES P388 IC₅₀ IC₅₀ IC₅₀ Neopeltolide (I) 1.17 nM 5.1 nM 0.56 nM

EXAMPLE 3 Antifungal Activity of Neopeltolide (I)

[0056] A. Effects of Neopeltolide on In Vitro Growth of Candida albicans

[0057] The antifungal activity of neopeltolide against Candida albicans(American Type Culture Collection strain 44506) was determined throughthe use of both agar diffusion and broth dilution assays.

[0058] Agar diffusion assays were performed using Sabouraud Dextroseagar plates seeded with the test microbe at 10⁵/ml. Disks (6.35 mm,Schliecher and Schuell) were impregnated with 25 μg of neopeltolide,allowed to dry and then placed on the agar surface. After incubation at37° C. for 24 hours the zones of growth inhibition were determined. Acontrol disk containing 100 IU nystatin was run on the same plate as theneopeltolide: the zone of inhibition was 28 mm.

[0059] Broth dilution assays were performed as standard 96-wellmicrotiter assays in a total volume of 501 μl with a seeding density of10³ cells/ml. The concentration range for neopeltolide ranged from 5 to0.4 μg/ml as two-fold dilutions. Plates were incubated at 37° C. for 24hours at which time the Minimum Inhibitory Concentration (MIC) wasdetermined as the lowest concentration in the test range that completelyinhibited growth of C. albicans. 5-fluorocytosine was used as anantifungal control for the assay: the MIC for 5-fluorocytosine was 0.62μg/ml.

[0060] Results: Neopeltolide I shows a growth inhibitory zone of 17 mmwhen tested at a concentration of 25 μg/disk in the C. albicans diskdiffusion assay. The Minimum inhibitory concentration (MIC) ofneopeltolide against C. albicans is 0.625 μg/ml.

EXAMPLE 4 Formulation and Administration

[0061] The compounds of the invention are useful for variousnon-therapeutic and therapeutic purposes. It is apparent from thetesting that the compounds of the invention are effective for inhibitingcell growth. Because of the antiproliferative properties of thecompounds, they are useful to prevent unwanted cell growth in a widevariety of settings including in vitro uses. They are also useful asstandards and for teaching demonstrations. As disclosed herein, they arealso useful prophylactically and therapeutically for treating cancercells in animals and humans.

[0062] Therapeutic application of the new compounds and compositionscontaining them can be accomplished by any suitable therapeutic methodand technique presently or prospectively known to those skilled in theart. Further, the compounds of the invention have use as startingmaterials or intermediates for the preparation of other useful compoundsand compositions.

[0063] The dosage administration to a host in the above indications willbe dependent upon the identity of the cancer cells, the type of hostinvolved, its age, weight, health, kind of concurrent treatment, if any,frequency of treatment, and therapeutic ratio.

[0064] The compounds of the subject invention can be formulatedaccording to known methods for preparing pharmaceutically usefulcompositions. Formulations are described in detail in a number ofsources which are well known and readily available to those skilled inthe art. For example, Remington's Pharmaceutical Science by E. W. Martindescribes formulations which can be used in connection with the subjectinvention. In general, the compositions of the subject invention will beformulated such that an effective amount of the bioactive compound(s) iscombined with a suitable carrier in order to facilitate effectiveadministration of the composition.

[0065] In accordance with the invention, pharmaceutical compositionscomprising, as an active ingredient, an effective amount of one or moreof the new compounds and one or more non-toxic, pharmaceuticallyacceptable carrier or diluent. Examples of such carriers for use in theinvention include ethanol, dimethyl sulfoxide, glycerol, silica,alumina, starch, and equivalent carriers and diluents.

[0066] To provide for the administration of such dosages for the desiredtherapeutic treatment, new pharmaceutical compositions of the inventionwill advantageously comprise between about 0.1% and 45%, and especially,1 and 15%, by weight of the total of one or more of the new compoundsbased on the weight of the total composition including carrier ordiluent. Illustratively, dosage levels of the administered activeingredients can be: intravenous, 0.01 to about 20 mg/kg;intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 to about100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about200 mg/kg, and preferably about 1 to 100 mg/kg; intranasal instillation,0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal(body) weight.

[0067] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the scope of the appended claims.

We claim:
 1. A compound having the following structural formula:

or a salt thereof.
 2. The compound, according to claim 1, having thefollowing formula:


3. The compound, according to claim 1, having the followingspectroscopic properties: Atom # ¹H (mult. J in Hz) ¹³C (mult)  1 —172.5 (s)  2 A 2.66 (dd J = 14.8, 4.3)  43.0 (t) B 2.33 (dd J = 14.5,10.8)  3 4.09 (m)  70.6 (d)  4 A 1.84 (m)  35.9 (t) B 1.53 (m)  5 5.21(m)  68.5 (d)  6 A 1.68 (m)  37.0 (t) B 1.54 (m)  7 3.58 (dd J = 10.4,9.8)  76.6 (d)  8 A 1.40 (m)  44.8 (t) B 1.29 (m)  9 1.45 (m)  32.0 (d)10 A 1.58 (m)  42.9 (t) B 1.14 (m) 11 3.63 (m)  76.5 (d) 12 A 1.90 (m) 40.5 (t) B 1.39 (m) 13 5.18 (m)  73.8 (d) 14 A 1.75 (m) B 1.53 (m) 37.5 (t) 15 AB 1.39 (2H m)  19.6 (t) 16 0.93 (3H t J = 7.3)  14.1 (q)17 0.99 (3H d J = 8.8)  25.9 (q) 11-OCH3 3.33 (3H s)  56.4 (q)  1′ —166.4 (s)  2′ 5.91 (bd J = 11.3) 121.3 (d)  3′ 6.35 (dt J = 11.3, 7.45)149.8 (d)  4′ AB 3.02 (2H q J = 7.45)  28.4 (t)  5′ AB 2.73 (2H t J =7.45)  26.0 (t)  6′ — 141.5 (s)  7′ 7.55 (bs) 135.3 (d)  8′ — 161.3 (s) 9′ 6.26 (bd J = 12.0) 115.8 (d) 10′ 6.05 (ddd J = 12.0, 6.0, 6.0) 138.5(d) 11′ AB 4.31 (2H bd J = 4.9)  40.6 (t) 12′ 158.9 (s) 12′-OCH3 3.67(3H s)  52.4 (q)


4. A method for inhibiting cellular proliferation, said methodcomprising administering to a patient in need of such treatment aneffective amount of a compound having the following structure:

or a salt of said compound.
 5. The method, according to claim 4, whereinsaid compound has the following structure:


6. The method, according to claim 4, wherein said compound has thefollowing copic properties: Atom # ¹H (mult. J in Hz) ¹³C (mult)  1 —172.5 (s)  2 A 2.66 (dd J = 14.8, 4.3)  43.0 (t) B 2.33 (dd J = 14.5,10.8)  3 4.09 (m)  70.6 (d)  4 A 1.84 (m)  35.9 (t) B 1.53 (m)  5 5.21(m)  68.5 (d)  6 A 1.68 (m)  37.0 (t) B 1.54 (m)  7 3.58 (dd J = 10.4,9.8)  76.6 (d)  8 A 1.40 (m)  44.8 (t) B 1.29 (m)  9 1.45 (m)  32.0 (d)10 A 1.58 (m)  42.9 (t) B 1.14 (m) 11 3.63 (m)  76.5 (d) 12 A 1.90 (m) 40.5 (t) B 1.39 (m) 13 5.18 (m)  73.8 (d) 14 A 1.75 (m) B 1.53 (m) 37.5 (t) 15 AB 1.39 (2H m)  19.6 (t) 16 0.93 (3H t J = 7.3)  14.1 (q)17 0.99 (3H d J = 8.8)  25.9 (q) 11-OCH3 3.33 (3H s)  56.4 (q)  1′ —166.4 (s)  2′ 5.91 (bd J = 11.3) 121.3 (d)  3′ 6.35 (dt J = 11.3, 7.45)149.8 (d)  4′ AB 3.02 (2H q J = 7.45)  28.4 (t)  5′ AB 2.73 (2H t J =7.45)  26.0 (t)  6′ — 141.5 (s)  7′ 7.55 (bs) 135.3 (d)  8′ — 161.3 (s) 9′ 6.26 (bd J = 12.0) 115.8 (d) 10′ 6.05 (ddd J = 12.0, 6.0, 6.0) 138.5(d) 11′ AB 4.31 (2H bd J = 4.9)  40.6 (t) 12′ 158.9 (s) 12′-OCH3 3.67(3H s)  52.4 (q)


7. The method, according to claim 4, wherein the cellular proliferationis associated with a condition selected from group consisting ofautoimmune disorders, inflammation, tumors and cancer.
 8. The method,according to claim 7, wherein the cancer is selected from the groupconsisting of breast cancer, colon cancer, cancer, CNS cancer, livercancer, lung cancer, leukemia, melanoma, ovarian cancer, uterine cancer,renal cancer, pancreatic cancer and prostate cancer.
 9. A pharmaceuticalcomposition comprising a compound having the following structure:

or a salt of said compound, wherein said composition further comprises apharmaceutically acceptable carrier.
 10. The pharmaceutical composition,according to claim 9, comprising a compound having the followingstructure:


11. The composition, according to claim 9, comprising a compound withthe following spectroscopic properties: Atom # ¹H (mult. J in Hz) ¹³C(mult)  1 — 172.5 (s)  2 A 2.66 (dd J = 14.8, 4.3)  43.0 (t) B 2.33 (ddJ = 14.5, 10.8)  3 4.09 (m)  70.6 (d)  4 A 1.84 (m)  35.9 (t) B 1.53 (m) 5 5.21 (m)  68.5 (d)  6 A 1.68 (m)  37.0 (t) B 1.54 (m)  7 3.58 (dd J =10.4, 9.8)  76.6 (d)  8 A 1.40 (m)  44.8 (t) B 1.29 (m)  9 1.45 (m) 32.0 (d) 10 A 1.58 (m)  42.9 (t) B 1.14 (m) 11 3.63 (m)  76.5 (d) 12 A1.90 (m)  40.5 (t) B 1.39 (m) 13 5.18 (m)  73.8 (d) 14 A 1.75 (m) B 1.53(m)  37.5 (t) 15 AB 1.39 (2H m)  19.6 (t) 16 0.93 (3H t J = 7.3)  14.1(q) 17 0.99 (3H d J = 8.8)  25.9 (q) 11-OCH3 3.33 (3H s)  56.4 (q)  1′ —166.4 (s)  2′ 5.91 (bd J = 11.3) 121.3 (d)  3′ 6.35 (dt J = 11.3, 7.45)149.8 (d)  4′ AB 3.02 (2H q J = 7.45)  28.4 (t)  5′ AB 2.73 (2H t J =7.45)  26.0 (t)  6′ — 141.5 (s)  7′ 7.55 (bs) 135.3 (d)  8′ — 161.3 (s) 9′ 6.26 (bd J = 12.0) 115.8 (d) 10′ 6.05 (ddd J = 12.0, 6.0, 6.0) 138.5(d) 11′ AB 4.31 (2H bd J = 4.9)  40.6 (t) 12′ 158.9 (s) 12′-OCH3 3.67(3H s)  52.4 (q)


12. A method for inhibiting fungal growth, said method comprisingapplying to a fungus a compound having the following structure:

or a salt of said compound.
 13. The method, according to claim 12,wherein said compound has the following structure:


14. The method, according to claim 12, wherein said compound has thefollowing spectroscopic properties: Atom # ¹H (mult. J in Hz) ¹³C (mult) 1 — 172.5 (s)  2 A 2.66 (dd J = 14.8, 4.3)  43.0 (t) B 2.33 (dd J =14.5, 10.8)  3 4.09 (m)  70.6 (d)  4 A 1.84 (m)  35.9 (t) B 1.53 (m)  55.21 (m)  68.5 (d)  6 A 1.68 (m)  37.0 (t) B 1.54 (m)  7 3.58 (dd J =10.4, 9.8)  76.6 (d)  8 A 1.40 (m)  44.8 (t) B 1.29 (m)  9 1.45 (m) 32.0 (d) 10 A 1.58 (m)  42.9 (t) B 1.14 (m) 11 3.63 (m)  76.5 (d) 12 A1.90 (m)  40.5 (t) B 1.39 (m) 13 5.18 (m)  73.8 (d) 14 A 1.75 (m) B 1.53(m)  37.5 (t) 15 AB 1.39 (2H m)  19.6 (t) 16 0.93 (3H t J = 7.3)  14.1(q) 17 0.99 (3H d J = 8.8)  25.9 (q) 11-OCH3 3.33 (3H s)  56.4 (q)  1′ —166.4 (s)  2′ 5.91 (bd J = 11.3) 121.3 (d)  3′ 6.35 (dt J = 11.3, 7.45)149.8 (d)  4′ AB 3.02 (2H q J = 7.45)  28.4 (t)  5′ AB 2.73 (2H t J =7.45)  26.0 (t)  6′ — 141.5 (s)  7′ 7.55 (bs) 135.3 (d)  8′ — 161.3 (s) 9′ 6.26 (bd J = 12.0) 115.8 (d) 10′ 6.05 (ddd J = 12.0, 6.0, 6.0) 138.5(d) 11′ AB 4.31 (2H bd J = 4.9)  40.6 (t) 12′ 158.9 (s) 12′-OCH3 3.67(3H s)  52.4 (q)


15. The method, according to claim 12, used to inhibit the spoilage offood or cosmetics.